This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In human populations, mutations in the NKX2-5 gene can lead to cardiac abnormalities, including AV block. Previously we have characterized a mouse model of Nkx2-5 haploinsufficiency, which leads to disease of the atrio-ventricular conduction system (AVCS). Specifically, a reduction in the numbers of peripheral Purkinje fibers was identified and complex abnormalities in the AV node were discovered. We hypothesize that abnormally low Nkx2-5 expression levels disrupt AV nodal morphogenesis. SA 1: To establish the ontogenic mechanism of the observed effects of Nkx2-5 haploinsufficiency on AVCS structure and function. I will undertake studies of the embryonic and postnatal AVCS of Nkx2-5+/- mice, to demonstrate that these deficits occur during development. SA 2: To selectively cause complete or partial loss of Nkx2-5 function in the AV node by crossing the c-GATA6/Cre transgenic mouse with an Nkx2-5-Flox line. The c-GATA6 enhancer directs expression specifically within the central AVCS and will allow the examination of the effects of homozygous or heterozygous AV nodal restricted knockout of Nkx2-5. SA 3: To test the hypothesis that extrinsic cardiac cell populations contribute to normal AV nodal development. I will use Wnt-1Cre and WT1Cre mice to trace the lineage contribution of neural crest cells and epicardial cells respectively, to AV nodal development. Novel 3D reconstructions show for the first time the normal development of the mouse AV node. My results indicate that the abnormal AV node in Nkx2-5+/- mice is indeed caused by a developmental insufficiency.